TULP2 deletion mice exhibit abnormal outer dense fiber structure and male infertility

Abstract Purpose Tulp2 (tubby‐like protein 2) is a member of the tubby protein family and expressed predominantly in mouse testis. Recently, it was reported that Tulp2 knockout (KO) mice exhibited disrupted sperm tail morphology; however, it remains to be determined how TULP2 deletion causes abnormal tail formation. Methods The authors analyzed male fertility, sperm morphology, and motility of two Tulp2 KO mouse lines that were generated using the conventional method that utilizes homologous recombination in embryonic stem (ES) cells as well as the clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) system. Furthermore, the authors observed the spermatogenesis of Tulp2 KO mice in more detail using scanning and transmission electron microscopy (SEM and TEM). Results Both mouse lines of Tulp2 KO exhibited male infertility, abnormal tail morphology, and impaired sperm motility. No overt abnormalities were found in the formation of the mitochondrial sheath in Tulp2 KO mice using the freeze‐fracture method with SEM. In contrast, abnormal outer dense fiber (ODF) structure was observed in Tulp2 KO testis with TEM. Conclusions TULP2 may play roles in the correct formation and/or maintenance of ODF, which may lead to abnormal tail morphology, impaired sperm motility, and male infertility.


| INTRODUC TI ON
Spermatozoa are highly specialized cells and consist of a head containing genetic information and a tail that moves vigorously to reach eggs. The sperm tail can be divided into three parts, midpiece, principal piece, and endpiece. 1,2 An axoneme, a motility apparatus that is composed of "9+2" microtubule arrangement, is found throughout the tail and each tail segment is classified according to the accessory structures surrounding the axoneme. The midpiece contains spirally arranged mitochondria called the mitochondrial sheath and outer dense fibers (ODFs) while the principal piece possesses the fibrous sheath and ODFs. The endpiece contains no accessory structures. Abnormal formation of these structures during spermatogenesis could lead to impaired sperm motility and male infertility.
Tubby family proteins are characterized by the tubby domain in the C-terminus and are evolutionarily conserved in animals and plants. 3,4 In mammals, there are five tubby family proteins, TUB, TULP1, TULP2, TULP3, and TULP4. TUB was identified first among tubby family proteins, whose mutation results in obesity, retinal degeneration, and hearing loss in mice. 5,6 Tulp1 and Tulp3 knockout (KO) mice have been generated as well with Tulp1 KO mice exhibiting retinal degeneration 7 and Tulp3 KO mice being embryonic lethal. 8 Further analyses indicate that TULP3 functions as an adapter protein for ciliary trafficking of membrane-associated proteins such as G protein-coupled receptors 9,10 and nephronspecific Tulp3 conditional KO mice exhibited cystic kidneys. 11 TULP1 is also suggested to be involved in intraflagellar transport in photoreceptor cells. 12 Unlike other TULP family proteins, Tulp2 is expressed predominantly in the mouse and human testes. 13,14 Tulp2 KO mice were generated recently and exhibited male infertility due to abnormal sperm tail formation. 14 In this study, we generated Tulp2 KO mice using not only the conventional method that utilizes homologous recombination in embryonic stem (ES) cells but also the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system and observed their defective tail formation in the testes using scanning and transmission electron microscopy (SEM and TEM).

| Animals
Mice were purchased from CLEA Japan, Inc. (Tokyo, Japan) or Japan

| Generation of Tulp2 KO mice using the CRISPR/Cas9 system
Knockout mice were generated using the CRISPR/Cas9 system, as described previously. 17 The guide RNAs (gRNAs) with fewer offtarget sites were designed with the online software CRISPRdirect. 18 The gRNA target sequences are shown in Supplementary Table S1.
KO mice were maintained on a B6D2 background. Primers used for genotyping are shown in Supplementary Table S1.

| Fertility analysis of Tulp2 KO lines
Sexually mature wild-type (WT) or Tulp2 KO male mice were caged individually with three seven-week-old female mice for at least five weeks. Male mice were removed after the mating period and females were kept for another three weeks to count the final litters. The numbers of pups and copulation plugs were counted every morning.

| Analysis of morphology and motility of spermatozoa
Spermatozoa from cauda epididymis were suspended in Toyoda, Yokoyama, Hoshi (TYH) medium 19 and placed on MAS-coated glass slides (Matsunami Glass) for morphology assessment using an Olympus BX53 microscope. For analyzing sperm motility, spermatozoa were incubated in TYH medium for 10 or 120 min and placed in glass chambers (Leja). Sperm motility was analyzed as previously described 20 using the CEROS II sperm analysis system (software version 1.5; Hamilton Thorne Biosciences).

| Ultrastructural analysis using transmission electron microscope (TEM)
Testis and cauda epididymis samples were prepared, as previously described. 21 The ultra-thin sections were observed using a JEM-1400plus electron microscope (JEOL) at 80 kV with a CCD Veleta 2K × 2K camera (Olympus).

| Ultrastructural analysis using scanning electron microscope (SEM)
Testis samples were prepared with the freeze-fracture method, as described previously. 21,22 The samples were observed with an S-4800 field emission scanning electron microscope (FE-SEM) (Hitachi).

| Statistical analyses
Statistical differences were determined using Welch's t-test by

| Generation of Tulp2 tm1/tm1 mice and the results of fertility test
To investigate the function of Tulp2 in vivo, we generated KO mice (Tulp1 tm1/tm1 mice) using homologous recombination in ES cells and subsequent generation of chimeric mice. Exon 7 (188 bp) of Tulp2, the longest coding exon among exons whose nucleotide length is not divisible by three, was replaced by a neomycin resistance gene cassette (neo) ( Figure 1A). Tulp2 tm1/tm1 mice were obtained by germline transmission and subsequent mating, which was confirmed by genomic PCR ( Figure 1B) using primers shown in Figure 1A. Tulp2 tm1/tm1 mice were viable and showed no overt abnormalities.
To analyze the fertility of Tulp2 tm1/tm1 males, the mice were caged with three WT females for more than five weeks. Although vaginal plugs were observed 19 times, no pups were obtained from Tulp2 tm1/tm1 males ( Figure 1C), indicating that Tulp2 is essential for male fertility.

| Analyses of sperm morphology and motility in Tulp2 tm1/tm1 mice
To analyze the cause of male infertility in Tulp2 tm1/tm1 males, we observed testis sections with a light microscope, but no apparent differences were found between WT and Tulp2 tm1/tm1 mice (Supplementary Figure S1). We then observed spermatozoa obtained from the cauda epididymis. In Tulp2 tm1/tm1 males, spermatozoa exhibited abnormal bending or coiling in the midpiece ( Figure 1D). Furthermore, motile spermatozoa were rarely observed in Tulp2 tm1/tm1 males ( Figure 1E) using a computer-assisted sperm analysis (CASA) system. These results suggest that male infertility in Tulp2 tm1/tm1 mice is caused by abnormal sperm morphology and motility.

| Generation and analyses of Tulp2 em1/em1 mice
It is possible that Tulp2 tm1/tm1 mice still generate functional TULP2 because of alteration in splicing patterns. To confirm that whole deletion of Tulp2 results in the same phenotypes as Tulp2 tm1/tm1 mice, we also generated KO mice (Tulp2 em1/em1 mice) using the CRISPR/ Cas9 system that has advanced remarkably in recent years. 23 We designed two gRNAs to delete almost the entire open reading frame of Tulp2 (Figure 2A). Ribonucleoprotein complexes that contain the gRNA and CAS9 were electroporated into 85 pronuclear embryos and 60 two-cell embryos were transferred into two pseudopregnant females the next day. Four out of nine pups had large deletions, and one male was caged with two WT females to obtain the F1 generation. Subsequent matings resulted in a KO mouse with an 8,420 bp deletion, which was confirmed by PCR ( Figure 2B) and Sanger sequencing ( Figure 2C). No overt abnormalities were found in the Tulp2 em1/em1 mice.
Tulp2 em1/em1 males were caged with three females for more than eight weeks. Although vaginal plugs were observed 37 times, no pups were obtained from Tulp2 em1/em1 males ( Figure 2D). Furthermore, as consistent with Tulp2 tm1/tm1 males, spermatozoa of Tulp2 em1/em1 mice exhibited abnormal morphology in the midpiece ( Figure 2E) and almost no motility with CASA ( Figure 2F). These results confirm that Tulp2 is essential for sperm tail formation, motility, and male fertility.

| Analyses of sperm morphology in the epididymis
To further analyze sperm morphological abnormalities in Tulp2 tm1/tm1 mice, we observed spermatozoa in the cauda epididymis using TEM. In the control, mitochondria were helically arranged in the midpiece ( Figure 3A). In contrast, the midpiece was disorganized with abnormal mitochondria localization and multiple axonemes enclosed in one plasma membrane in Tulp2 tm1/tm1 mice ( Figure 3A), which is consistent with coiling midpieces observed with light microscopy ( Figures 1D and 2E).
To analyze when abnormal midpiece bending or coiling emerged, we observed spermatozoa obtained from caput, corpus, or cauda

| Analyses of sperm morphology in the testis
Because abnormal midpieces were already observed in the caput epididymis ( Figure 3B,C), we analyzed mitochondrial sheath formation in testes using the freeze-fracture method with SEM. 21,22 Unexpectedly, mitochondrial sheath formation (alignment, interlocking, and compaction steps) in Tulp2 tm1/tm1 mice was comparable to that of control mice ( Figure 4A). We then observed spermiogenesis in testis using TEM (stage V and stage VIII). Consistent with the freeze-fracture method, mitochondrial sheath formation is comparable between Tulp2 +/tm1 and Tulp2 tm1/tm1 mice ( Figure 4B).
In contrast, we frequently observed an abnormal number of ODFs

| DISCUSS ION
By generating two mouse lines of Tulp2, we confirmed that Tulp2 is essential for male fertility. Further analyses indicate that Tulp2 KO mice exhibited abnormal tail formation and impaired sperm motility. Further morphological abnormalities of midpieces during the epididymal transit have also been reported in the KO mice of other genes such as Gk2 and Armc12. 21,22 Because the mitochondrial sheath was disrupted in the caput spermatozoa of Tulp2 tm1/tm1 mice ( Figure 3B,C), we observed the formation of mitochondrial sheath during spermatogenesis using the freeze-fracture method with SEM; however, no overt abnormalities were found in Tulp2 tm1/tm1 mice ( Figure 4A). In contrast, we observed numbers of extra ODFs with TEM ( Figure 4B). Because of this abnormal extra ODF, sperm tail structures including mitochondria sheath may be unstable and become disrupted during the later stages of spermatogenesis and sperm transit in the epididymis. Supporting this idea, extra ODFs and abnormal mitochondrial sheath structures were also observed in Cfap44 KO mice 24 and infertile patients with CFAP58 loss-of-function variants. 25 Because it is suggested that CFAP44 (cilia-and flagella-associated protein 44) plays a role in the connection between ODFs and the axoneme 24 and CFAP58 (cilia-and flagella-associated protein 58) influences ODF protein transportation, 25 TULP2 may be involved in these processes. TULP1 and TULP3 are thought to be involved in intraflagellar transport in cilia. [9][10][11][12] Because sperm flagella possess similar axonemal structures to cilia, it is tempting to hypothesize that TULP2 plays roles in intraflagellar transport of molecules that are involved in the correct ODF formation. However, a recent study indicates that TULP2 could not rescue impaired intraflagellar transport completely in Tulp3 KO RPE-1 cells although TUB could rescue the phenotype, 26 suggesting that TULP2 may not be involved in intraflagellar transport. However, it is still possible that TULP2 functions in intraflagellar transport of sperm flagella in a mechanism different from TULP3 in RPE-1 cells. It is also possible that TULP2 functions as a transcription factor as suggested by the function of TUB 27 or functions as an RNA-binding protein as recently suggested with the analyses of Tulp2 KO mice, 14 which impairment may lead to abnormal transcription and/or translation of molecules associated with ODFs. Although Cfap58 or Cfap44 was not listed, a recent study detected 1446 differentially expressed genes between WT and Tulp2 KO testes using RNA sequencing, some of which were related to the cytoskeleton. 14 In summary, we reveal that mitochondrial sheath formation is not disrupted, but ODF structure is abnormal in Tulp2 KO mice, which may lead to impaired sperm motility and male infertility. Further analysis of TULP2 may provide a better understanding of the mechanism that regulates the sperm tail formation and may lead to better treatment for male infertility.

ACK N OWLED G EM ENTS
We thank the Department of Experimental Genome Research and Animal Resource Center for Infectious Diseases for creating F I G U R E 4 Observation of sperm morphology in the testis. (A) Formation of the mitochondrial sheath during spermatogenesis observed with scanning electron microscopy (SEM). Spherical mitochondria align around the axoneme (left), change their shape in the mitochondrial interlocking step (middle), and form the mitochondrial sheath in the mitochondrial compaction step (right). Wild-type (WT) and Tulp2 +/tm1 mice were used as controls. (B) Observation of spermatids with transmission electron microscopy (TEM). Abnormal extra outer dense fibers (ODFs) are highlighted with white arrowheads. Higher magnification images of the boxed areas are shown to the right